Technical Development Project 1: Measuring the Immunome: Genomic Approaches to B Cell Repertoire. In this project, we propose to develop new technological applications of next generation DNA sequencing, high throughput PCR chips, and microfluidic single cell processors to perform systemic analyses of the human B cell repertoire. Using B cells purified from human peripheral blood, we will apply these technologies to try to understand the effects of three phenomena on immune repertoire: aging, genetic background and vaccination history. Four groups of subjects will be studied: 1. age groups - children, young adults and elderly;2. genetic background - identical twins vs fraternal twins vs not-related subjects;3. vaccination history - pre, post and multiple immunizations;4. vaccination type - the trivalent inactivated influenza vaccine (TIV) and the live attenuated influenza vaccine (LAIV). This study will provide the first comprehensive and systematic measurements of B cell repertoire at three different anatomic levels, V/D/J/C exon usage, immunoglobulin (Ig) gene deep sequencing, and single cell gene expression analysis. Our specific aims are: Aim 1. Develop a quick and quantitative assay to measure the V, D J, and C exon usage landscape. Aim 2. Develop a high throughput sequencing protocol to perform deep sequencing of Ig heavy chain gene diversity. Aim 3. Develop a microfluidic parallel processor for single cell analysis of B cell repertoire. Aim 4. Dissect the influences of aging, genetic background, and vaccination history on the human B cell repertoire.